Antifungal drug resistance is a significant clinical problem, and antifungal agents that can evade resistance are urgently needed. In infective niches, resistant organisms often co-existed with sensitive ones, or a subpopulation of antibiotic-susceptible organisms may evolve into resistant ones during antibiotic treatment and eventually dominate the whole population. In this study, we established a co-culture assay in which an azole-resistant Candida albicans strain was mixed with a susceptible strain labeled with green fluorescent protein to mimic in vivo conditions and screen for antifungal drugs. Fluconazole was used as a positive control to verify the validity of this co-culture assay. Five natural molecules exhibited antifungal activity against both susceptible and resistant C. albicans. Two of these compounds, retigeric acid B (RAB) and riccardin D (RD), preferentially inhibited C. albicans strains in which the efflux pump MDR1 was activated. This selectivity was attributed to greater intracellular accumulation of the drugs in the resistant strains. Changes in sterol and lipid compositions were observed in the resistant strains compared to the susceptible strain, and might increase cell permeability to RAB and RD. In addition, RAB and RD interfered with the sterol pathway, further aggregating the decrease in ergosterol in the sterol synthesis pathway in the MDR1-activated strains. Our findings here provide an alternative for combating resistant pathogenic fungi.
ATP-Binding Cassette Transporters ; genetics ; metabolism ; Antifungal Agents ; chemistry ; metabolism ; pharmacology ; Azoles ; pharmacology ; Biosynthetic Pathways ; drug effects ; genetics ; Candida albicans ; chemistry ; drug effects ; metabolism ; Cell Membrane ; chemistry ; metabolism ; Coculture Techniques ; Drug Resistance, Fungal ; drug effects ; Ergosterol ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Lipids ; chemistry ; Molecular Structure ; Permeability ; Phenyl Ethers ; chemistry ; metabolism ; pharmacology ; Sterols ; chemistry ; metabolism ; Stilbenes ; chemistry ; metabolism ; pharmacology ; Triterpenes ; chemistry ; metabolism ; pharmacology

OBJECTIVETo investigate the effect of c-Met inhibitor cabozantinib (XL-184) in inhibiting Listeria monocytogenes (LM) from invading Caco-2 cells to reduce the cell injury.

METHODSThe cell invasion capacity of LM was assayed in Caco-2 cells incubated with different doses of XL-184 for different durations. Caco-2 cells incubated with XL-184 were seeded on the upper room of the transwell chamber, and the cell monolayer was exposed to LM infection followed by addition of horseradish peroxidase (HRP). The trans-epithelial electric resistance (TEER), HRP concentration and LM colony-forming unit (CFU) were measured in the cell monolayer. Fluorescent staining was used to evaluate the cell viability, and LDH release from the cells was examined to assess the changes in cell membrane permeability.

RESULTSXL-184 significantly decreased LM invasion rate in Caco-2 cells in a dose- and time-dependent manner (P=0.000), and this effect was enhanced by co-incubation of the cells with ampicillin (P<0.05). In the cell membrane permeability assay in the monolayer cells, XL-184 markedly inhibited LM-induced reduction of TEER (P<0.05) and significantly suppressed LM-induced enhancement of cell membrane permeability shown by reduced HRP concentration and LM count in the lower chamber (P=0.000). The cells infected with LM showed significantly lowered cell viability, which was rescued by XL-184 (P<0.01); XL-184 also dose-dependently reduced LDH release from the cells (P<0.05).

CONCLUSIONSXL-184 can suppress LM invasion in Caco-2 cells to reduce the cell injury, suggesting its value as a promising candidate agent for prevention and treatment of LM infections.

Anilides ; pharmacology ; Caco-2 Cells ; Cell Membrane Permeability ; drug effects ; Cell Survival ; Humans ; Listeria monocytogenes ; drug effects ; Pyridines ; pharmacology

To investigate the influence of the difference enhancers on the transport mechanism of chlorogenic acid (CGA) across Caco-2 cells model, a RP-HPLC method was adopted to detect the concentrations of CGA. At the concentrations of 20 to 80 microg x mL(-1), the difference of absorption rate constants (K(a)) was not statistically significant. At the concentrations of 40 and 20 microg x mL(-1), the ratios of apparent permeability coefficients (P(app)) of the apical to basolateral and the basolateral to apical were 1.14 and 1.18, respectively. With the effect of enhancers K(a) and P(app) increased, the absorption half-life (T1/2) decreased. CGA passed through the Caco-2 cell membrane mainly by passive transport. It showed that monocarboxylic acid transporter (MCT) could be involved in the across membrane transport process of CGA. Borneol had no effect on the cell membrane transport processes. The order of increasing absorption of CGA caused by the enhancers was sodium lauryl sulphate > sodium taurocholate > carbomer.
Absorption ; Acrylic Resins ; pharmacology ; Caco-2 Cells ; Cell Membrane Permeability ; drug effects ; Chlorogenic Acid ; pharmacokinetics ; Humans ; Sodium Dodecyl Sulfate ; pharmacology ; Taurocholic Acid ; pharmacology

The microstructure of cationic cyclopeptide (TD-34) treated Caco-2 cell membrane was observed, and we discussed the relationship between membrane structure and insulin transmembrane permeability. Atomic force microscope (AFM) was used to observe living cell membrane in air condition and tapping mode. Results showed that the surface of Caco-2 cell membrane treated with TD-34 lost its smoothness and nearly doubled its roughness. Apparent permeability coefficients (P(app)) of insulin in Caco-2 cell monolayers increased 2.5 times. In conclusion, AFM can be used to observe microstructure of cationic cyclopeptide treated cell membrane and cationic cyclopeptide enhanced insulin delivery across Caco-2 cell membrane by increasing membrane fluidity.
Caco-2 Cells ; Cations ; Cell Membrane ; drug effects ; Cell Membrane Permeability ; drug effects ; Humans ; Insulin ; metabolism ; Membrane Fluidity ; drug effects ; Microscopy, Atomic Force ; Peptides, Cyclic ; pharmacology

Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca(2+)-dependent manner. In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca(2+) ([Ca(2+)]i) via Ca(2+) influx, Ca(2+) mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca(2+) via multiple mechanisms in beta-cells from both diabetic db/db mice and non-diabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca(2+)]i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca(2+) concentration in the ER, a compensatory up-regulation of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and a reduction in depolarization-evoked Ca(2+) influx. As a result, the patterns of glucose-stimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.
Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Membrane Permeability ; drug effects ; Cells, Cultured ; Down-Regulation ; drug effects ; Endoplasmic Reticulum ; metabolism ; Glucose ; pharmacology ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Obese ; Potassium Chloride ; pharmacology ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sodium-Calcium Exchanger ; metabolism ; Thapsigargin ; pharmacology ; Up-Regulation ; drug effects

OBJECTIVETo study the mechanism of the reducing mechanism of hepatotoxicity induced by ethyl acetate fractions of Kansui Radix stir-baked with vinegar in mice.

METHODMice with normal ICR were orally administered with ethyl acetate fractions of Kansui Radix and Kansui Radix stir-baked with vinegar. Their blood and liver homogenate were collected to detect the level of AST, ALT, LDH, SOD, activities of Na(+) -K(+) -ATPase and Ca(2+) -Mg(2+) -ATPase, GSH and MDA. Liver tissues were collected for HE staining and morphological observation under light microscope.

RESULTAccording to the results of pathological sections, compared with the control group, all of Kansui groups showed a significant increase in the hepatic tissues injury (P < 0.01). Compared with Kansui groups, all of vinegar-baked groups showed a significant decrease in the hepatic tissues injury (P < 0.01). Compared with the control group, all of Kansui groups showed a significant increase in ALT, AST and LDH (P < 0.05, P < 0.001) in serum and hepatic tissues, and significantly decrease in the activity of SOD (P < 0.001) and the content of GSH. They also showed a significant increase in MDA (P < 0.001) and a significant decrease in the level of Na(+) -K(+) -ATPase and Ca(2+) -Mg(2+) -ATPase (P < 0.01) in hepatic tissues, with a certain dose-effect relationship. Compared with all of Kansui groups, all of vinegar-baked groups showed a significant decrease in ALT, AST and LDH (P < 0.05, P < 0.001), and a notable increase in SOD (P < 0.001) and GSH in serum and hepatic tissues. They also showed a remarkable decrease in MDA (P < 0.001), and a significant increase in the level of Na(+) -K(+) -ATPase and Ca(2+) - Mg(2+) -ATPase (P < 0.01) in hepatic tissues, with a certain dose-effect relationship.

CONCLUSIONBeing stir-baked with vinegar can significantly reduce the hepatotoxicity of Kansui Radix. Its mechanism may be related to the reduction of the effect of Kansui Radix on the permeability of hepatic tissues cell membranes and the oxidative injury.

Acetic Acid ; Animals ; Cell Membrane Permeability ; drug effects ; Euphorbia ; toxicity ; Liver ; drug effects ; metabolism ; pathology ; Mice ; Oxidation-Reduction ; Plant Extracts ; toxicity

An experiment was carried out to study the allelopathic effects of Coptis chinensis fibrous root extracts (CRE) on the germination and seedling growth of Vicia faba and Pisum sativum in order to alleviate the allelopathic effects and increase land productivity. The seeds of both garden pea (P. sativum) and broad been (V. faba) were germinated in CRE solution of various concentrations, the germination rate, seedling growth and related physiological indexes were measured. The result indicated that there were no significant effects of CRE in low concentrations on seed germination, including both the rate and index, and seed vitality and membrane permeability. With the increment of CRE concentrations, however, the high seed membrane permeability and germination inhibition were observed. For example, the germination rates were reduced by 23.4% (P. sativum) and 9.5% (V. faba), respectively, in CRE solution with 800 mg . L-1. Simultaneously, soluble sugars and the free amino acids in the seeds were lower than those in the control (without CRE) after soaking seeds in CRE solutions. In addition, the seedling growth and nitrate reductase activity were stimulated by CRE at low concentrations in contrast to high concentrations which behaved otherwise and inhibited the nutrient utilization in endosperm. Therefore, the large amount of allelochemicals released from the roots and remains of C. chinensis in soils could inhibit the seed germination and seedling growth of legumes, which may lead to decrease even fail crop yields after growing this medical plant.
Amino Acids ; metabolism ; Cell Membrane Permeability ; drug effects ; Coptis ; chemistry ; Dose-Response Relationship, Drug ; Germination ; drug effects ; Nitrate Reductase ; metabolism ; Peas ; drug effects ; metabolism ; physiology ; Pheromones ; isolation & purification ; pharmacology ; Plant Extracts ; isolation & purification ; pharmacology ; Plant Roots ; chemistry ; Seedlings ; drug effects ; growth & development ; Vicia faba ; drug effects ; metabolism ; physiology

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.
Animals ; Cell Membrane Permeability ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Cells, Cultured ; Gene Knockdown Techniques ; Mice ; Mice, Knockout ; Podocytes ; drug effects ; physiology ; Puromycin Aminonucleoside ; pharmacology ; TRPC Cation Channels ; genetics ; metabolism

Baicalein (BAI) is an effective bactericide. The antibacterial activity and mechanism experiments were carried out by determining conductivity and content of macromolecules of membrane penetrability, the oxidative respiratory metabolism and protein synthesis changes and the inhibition of DNA topoisomerase activities. Electrical conductivity and the number of large molecules of BAI increased 2.48% and 1.8%, respectively, than that of the control. However, the membrane integrity did not destroyed by BAI directly. With BAI treatment, inhibition rates of activities for SDH and MDH were 56.2% and 57.4%, respectively, demonstrating that BAI could inhibit cell respiratory. After treated with BAI for 20 h, the total soluble content of proteins decreased by 42.83%. Moreover, the activities of DNA topoisomerase I and II were inhibited completely by 0.2 mmol x L(-1) BAI. These results indicated that BAI had obvious antibacterial activity on Staphylococcus aureus. The mechanism is that it could affect bacterial membrane penetrability, inhibit protein synthesis and influence SDH, MDH and DNA topoisomerase I and II activities to exert its antibacterial functions.
Anti-Bacterial Agents ; isolation & purification ; pharmacology ; Bacterial Proteins ; metabolism ; Cell Membrane Permeability ; drug effects ; DNA Topoisomerases, Type I ; metabolism ; DNA Topoisomerases, Type II ; metabolism ; Flavanones ; isolation & purification ; pharmacology ; Malate Dehydrogenase ; metabolism ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Scutellaria baicalensis ; chemistry ; Solubility ; Staphylococcus aureus ; cytology ; drug effects ; metabolism ; Succinate Dehydrogenase ; metabolism

To construct, express, purify and identify the Myc-R9-EGFP fusion protein and validate its transduction activity in the cultured porcine embryo fibroblasts. cDNA of pig c-Myc gene was amplified by RT-PCR with specific primers of 9 arginine (R9) from the primordial genital ridges and inserted into prokaryotic expression vector pET-28a-EGFP. After DNA sequencing confirmation, the recombinant plasmid was then transformed into BL21 (Escherichia coli) strain. After IPTG induction, the target fusion protein was efficiently induced to express, successfully purified by Novagen His-Bind kit, identified by SDS-PAGE and Western blotting. Finally, its high transduction activity in the porcine embryo fibroblasts was validated. The purified Myc-R9-EGFP fusion protein and the validation of its transduction activity in fibroblasts have provided an experimental foundation for further studies on the biological characterization of Myc protein, and soundly facilitated the further study of establishing pig induced pluripotent stem cells by recombinant protein.
Animals ; Arginine ; genetics ; metabolism ; Cell Line ; Cell Membrane Permeability ; drug effects ; Escherichia coli ; genetics ; metabolism ; Fibroblasts ; cytology ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacokinetics ; Swine